Canadian journal of microbiology最新文献

Conjugation is a complex phenomenon involving multiple plasmid, bacterial, and environmental factors. Here we describe an IncI1 plasmid encoding multidrug antibiotic resistance to aminoglycosides, sulfonamides, and third-generation cephalosporins. This plasmid is widespread geographically among animal, human, and environmental sectors. We present data on the transmissibility of this plasmid from Salmonella enterica ser. Kentucky into 40 strains of S. enterica (10 strains each from serovars Enteritidis, Heidelberg, Infantis, and Typhimurium). Thirty seven out of 40 strains were able to take up the plasmid. Rates of conjugation were variable between strains ranging from 10^-8 to 10^-4. Overall, serovars Enteritidis and Typhimurium demonstrated the highest rates of conjugation, followed by Heidelberg, and then Infantis. No relationships were observed between the recipient cell surface and rate of conjugation. Recipient cell numbers correlated positively with conjugation rate and strains with high conjugation rates had marginally but significantly higher growth parameters compared to strains that took up the plasmid at lower frequencies. Environmental conditions known to impact cell growth, such as temperature, nutrient availability, and the presence of antibiotics, had a modulating effect on conjugation. Collectively, these results will further understanding of plasmid transmission dynamics in Salmonella, which is a critical first step towards the development of mitigation strategies.

Rapidly developing microbial resistance to existing antimicrobials poses a growing threat to public health and global food security. Current chemical-based treatments target cells by inhibiting growth or metabolic function, but their effectiveness is diminishing. To address the growing antimicrobial resistance crisis, there is an urgent need for innovative therapies. Conjugative plasmids, a natural mechanism of horizontal gene transfer in bacteria, have been repurposed to deliver toxic genetic cargo to recipient cells, showing promise as next-generation antimicrobial agents. However, the ecological risks posed by unintended gene transfer require robust biocontainment strategies. In this study, we developed inducible conjugative plasmids to solve these challenges. Utilizing an arabinose-inducible promoter, we evaluated 13 plasmids with single essential gene deletions, identifying trbC and trbF as strong candidates for stringent regulation. These plasmids demonstrated inducibility in both cis and trans configurations, with induction resulting in up to a 5-log increase in conjugation efficiency compared to uninduced conditions. Although challenges such as reduced conjugation efficiency and promoter leakiness persist, this work establishes a foundation for the controlled transfer of plasmids, paving the way for safer and more effective antimicrobial technologies.

Fungal pathogens significantly impact human health, agriculture, and ecosystems, with infections leading to high morbidity and mortality, especially among immunocompromised individuals. The increasing prevalence of antifungal resistance (AFR) exacerbates these challenges, limiting the effectiveness of current treatments. Identifying robust biomarkers associated AFR could accelerate targeted diagnosis, shorten decision time for treatment strategies, and improve patient health. This paper examines traditional avenues of AFR biomarker detection, contrasting them with the increasingly effective role of machine learning (ML) in advancing diagnostic and therapeutic strategies. The integration of ML with technologies such as mass spectrometry, molecular dynamics, and various omics-based approaches often results in the discovery of diverse and novel resistance biomarkers. ML's capability to analyse complex data patterns enhances the identification of resistance biomarkers and potential drug targets, offering innovative solutions to AFR management. This paper highlights the importance of interdisciplinary approaches and continued innovation in leveraging ML to combat AFR, aiming for more effective and targeted treatments for fungal infections.

Botrytis cinerea is a necrotrophic fungal pathogen responsible for grey mold disease, causing significant crop losses globally. Effective genetic manipulation of this pathogen is crucial for understanding its biology and developing better disease management strategies. However, current transformation methods typically use conidia spores or protoplasts, processes that are labor-intensive, inefficient, and often yield inconsistent results. We developed a novel Agrobacterium-mediated transformation protocol that utilizes B. cinerea mycelia, eliminating the need for sporulation or protoplast generation and simplifying genetic manipulation. Using a newly constructed binary expression vector encoding green fluorescent protein and hygromycin resistance, we transformed four different B. cinerea strains. All transformations resulted in stable integration and robust green fluorescent protein expression, confirmed by quantitative polymerase chain reaction and confocal microscopy. Although transformants exhibited altered colony morphology compared to wild-type strains, they remained viable and stably expressed the integrated transgene, supporting the method's utility for genetic studies in B. cinerea. This streamlined method provides a reliable, efficient, and scalable approach for genetic studies in B. cinerea, significantly enhancing fungal functional genomics research and plant pathology investigations.

The opportunistic human pathogen Pseudomonas aeruginosa exhibits high pathogenicity and antimicrobial resistance, largely due to its ability to form robust biofilms. In addition to the exopolysaccharides Psl and Pel, extracellular DNA (eDNA) is an important matrix component in P. aeruginosa PAO1 biofilms. It has been shown previously that eDNA is involved in biofilm initiation and integrity as well as antibiotic resistance; however, its involvement in resistance to oxidative stressors such as the widely used disinfectant sodium hypochlorite (NaOCl) is less explored. Here, we examined the function of eDNA in NaOCl resistance of P. aeruginosa PAO1 biofilms. Using different biofilm assays in combination with a PAO1 ∆pslA pelF double mutant, which lacks the exopolysaccharides Psl and Pel, and a Tn-bfmR mutant, which exhibits increased eDNA amounts in biofilms, we were able to show that eDNA contributes to NaOCl resistance in P. aeruginosa PAO1 biofilms, in particular when exopolysaccharides are absent. Interestingly, NaOCl was more effective after DNase treatment against ∆pslA pelF biofilms. These findings indicate that the protective function of eDNA in biofilm resistance is matrix composition-dependent and becomes more pronounced in the absence of Psl and Pel.

Ingestion of antibiotic-resistant bacteria following antibiotic treatments may lead to the transfer of antimicrobial resistance genes (ARGs) within a disturbed gut microbiota. However, it remains unclear whether and how microbes present in food matrices influence ARG transfer. Thus, a previously established mouse model, which demonstrated the conjugative transfer of a multi-drug resistance plasmid (pIncA/C) from Salmonella Heidelberg (donor) to Salmonella Typhimurium (recipient), was used to assess the effects of food-borne microbes derived from fresh carrots on pIncA/C transfer. Mice were pre-treated with ampicillin, streptomycin, sulfamethazine, or left untreated as a control to facilitate bacterial colonization. Contrary to previous findings where high-density colonization of the donor and recipient bacteria occurred in the absence of food-borne microbes, the presence of these microbes resulted in a low abundance of S. Typhimurium and no detection of S. Typhimurium transconjugants in the fecal samples from any of the mice. However, in mice pre-treated with streptomycin, a significant reduction in microbial species richness allowed for the significant enrichment of Enterobacteriaceae and pIncA/C transfer to bacteria from the genera Escherichia, Enterobacter, Citrobacter, and Proteus. These findings suggest that food-borne microbes may enhance ARG dissemination by influencing the population dynamics of bacterial hosts within a pre-disturbed gut microbiome.

Mango (Mangifera indica L.) is famous for its flavor, aroma, and nutritional value. However, anthracnose caused by Colletotrichum is the most destructive postharvest disease of mango, causing significant economic losses. This study aimed to identify and characterize Colletotrichum species associated with mango anthracnose in Vietnam and evaluate their pathogenicity and cross-infection potential. Through examination of colony characteristics, conidia, and appressorial morphology, along with phylogenetic analysis based on the ITS region and five genetic markers (gapdh, act, tub2, chs-1, and cal), five isolates were classified into three distinct species: C. asianum (MH32, MH24, and MC76), C. fructicola (MC32), and C. laticiphilum (MC81). Notably, C. fructicola and C. laticiphilum are the two species identified for the first time on mangoes in Vietnam. Pathogenicity tests demonstrated that C. fructicola MC32 and C. laticiphilum MC81 caused anthracnose in mango, banana, guava, and tomato. Among the C. asianum isolates, differences in aggressiveness were observed: isolate MH32 caused anthracnose on mango, banana, guava, and tomato; isolate MC76 caused anthracnose on mango, banana, and tomato; and isolate MH24 affected only mango and tomato.

The Canadian Genomics Research and Development Initiative for Antimicrobial Resistance (GRDI-AMR) uses a genomics-based approach to understand how health care, food production and the environment contribute to the development of antimicrobial resistance. Integrating genomics contextual data streams across the One Health continuum is challenging because of the diversity in data scope, content and structure. To better enable data harmonization for analyses, a contextual data standard was developed. However, development of standards does not guarantee their use. Implementation strategies are critical for putting standards into practice. This work focuses on the development of implementation strategies to better operationalize data standards across the Canadian federal genomics ecosystem. Results include improved understanding of complex data models that can create challenges for existing systems. Technical implementation strategies included spreadsheet-based solutions, new exchange formats, and direct standards integration into new databases. Data curation exercises highlighted common data collection and sharing issues, which informed improved practices and evaluation procedures. These new practices are contributing to improved data quality and sharing within the GRDI-AMR consortium as evidenced by publicly available datasets. The implementation strategies and lessons learned described in this work are generalizable for other standards and can be applied more broadly within other initiatives.

Agricultural practices, specifically the use of antibiotics and other biocides, have repercussions on human, animal, and plant health. The aim of this study was to evaluate the levels of Enterobacteriaceae and Enterococcus, as antibiotic-resistant marker bacteria, in various matrices across the agro-ecosystem of an antibiotic-free swine farm in Quebec (Canada), namely pig feed, feces, manure, agricultural soil, water and sediment from a crossing stream, and soil from nearby forests. Samples were collected in fall 2022, spring and fall 2023, and spring 2024. All samples were subjected to counts of total, cefotaxime-, and ciprofloxacin-resistant Enterobacteriaceae as well as total and vancomycin-resistant Enterococcus spp. The frequency of total and cefotaxime-resistant Enterobacteriaceae along with the vancomycin-resistant Enterococcus decreased with age in pig feces, from weaning to the end of the fattening period. High proportions of the Enterobacteriaceae recovered from feces and environmental samples were resistant to cefotaxime. Application of manure on fields contributed a significant input of Enterococcus, but those resistant to vancomycin were under the detection limit. This study shows the prevalence of antibiotic-resistant bacteria in a farm agro-ecosystem even without the administration of antibiotics to the animals and highlights the complexity of components influencing antimicrobial resistance in the environment.

Superficial fungal infections, mainly caused by dermatophytes, are a global public health issue. We evaluated the antifungal activity of six β-amino-ketones against Trichophyton rubrum, a leading agent of superficial mycoses. Among them, 3-(morpholin-4-yl)-1-phenylpropan-1-one (AB1) showed the most potent effect, with a minimum inhibitory concentration (MIC) of 7.81 µg/mL against the T. rubrum reference strain and fungicidal activity against clinical isolates, as demonstrated by minimum fungicidal concentration assays. AB1 was effective against both conidia and hyphae of T. rubrum, while showing limited activity against Candida albicans and the bacteria Escherichia coli and Staphylococcus aureus. Mechanistic studies suggest AB1 targets the fungal plasma membrane, possibly via ergosterol interactions, supported by increased MICs in ergosterol-rich conditions and membrane integrity assays. Confocal microscopy revealed morphological alterations in AB1-treated hyphae, indicative of membrane damage. Transmission electron microscopy confirmed cytoplasmic disorganization and membrane disruption at subinhibitory concentrations. Toxicological assays showed moderate cytotoxicity in human fibroblasts (IC₅₀ = 37.75 µg/mL) and no toxicity in Galleria mellonella larvae at high doses. These findings highlight AB1 as a promising antifungal candidate against Trichophyton spp., with the potential benefit of reduced impact on the host microbiota compared to broad-spectrum antimicrobials.